PI: Gelis, Ioannis Project Summary Hsp90 in cohort with other proteins termed cochaperones, forms a molecular machinery that controls the conformational maturation of a diverse array of substrates or clients. The Hsp90-mediated chaperone cycle occurs at a late stage of protein folding to increase client stability and/or to elicit fine structural changes which facilitate ?partner? binding and/or result in client activation. It begins with client recognition by a recruiter cochaperone and subsequent client transfer to Hsp90. The cochaperone is then released and client remodeling and release from Hsp90 is accompanied by large conformational changes of Hsp90. Progression between these sequential steps is finely modulated by the presence of other cochaperones, the identity of the nucleotide bound to Hsp90 and post-tranlational modifications. At a molecular level, the interaction of Hsp90 with its cochaperones and the conformational changes associated with its ATPase cycle have been characterized at atomic resolution. Although these studies have contributed significantly to our overall understanding of the function of the chaperone, the molecular mechanism by which substrates become engaged into the chaperone cycle remains poorly understood. During the first step of Hsp90-mediated kinase chaperoning, Cdc37 is the ubiquitous substrate recruiter cochaperone associated with Hsp90 to confer kinase specificity. Despite its central role in kinase chaperoning, little is known about the molecular mechanism by which Cdc37 recognizes protein kinases, sorts Hsp90-dependent from Hsp90-independent kinases and delivers them to Hsp90. We will use a multidisciplinary approach, where high resolution structural and dynamic information from NMR spectroscopy, will be combined with thermodynamics and kinetics of association, as well as, with in vivo functional assays, to elucidate the molecular mechanism that allows Cdc37 to control kinase entry into the Hsp90 chaperone cycle. The specific aims are designed to address the following questions (1) what are the structural and dynamic properties of Cdc37; (2) how does Cdc37 recognizes the catalytic domain of protein kinases; and (3) how Cdc37 differentiates between Hsp90-dependent and Hsp90-independent kinases?